Purpose: To identify and differentiate between different types of blood cells.

Principle: Leishman stain is a mixture of methylene blue dye (blue in colour) and eosin dye (pink in colour). Methylene blue is basic, while eosin dye is acidic. As a result, methylene blue binds to acidic components of the cell, such as nucleic acids in the nucleus, and to acidic granules present in certain cells. Eosin dye, on the other hand, binds to basic components of the cell, including cytoplasmic granules. When a stained blood smear is observed under a microscope, differences in the shape, size, and colour of the stained nuclei and cytoplasmic granules allow different types of blood cells to be identified.

Protocol:

  1. Using fresh blood, make a smear on a clean, grease-free glass slide.
  2. Place a drop of blood near one end of the slide.
  3. Hold another slide at a 45° angle and place it just behind the drop.
  4. Allow the blood to spread along the edge of the second slide, then move it smoothly across the first slide in one motion.
  5. A thin smear of blood should form on the slide.
  6. Allow the smear to air dry completely.
  7. Dip the slide into a coupling jar containing Leishman’s stain for 2 minutes.
  8. Remove the slide and dip it into another coupling jar containing distilled water for 1 minute.
  9. Rinse briefly by dipping the slide again in distilled water and remove.
  10. Clean the back of the slide and observe under the microscope using immersion oil at 100× magnification.

Characterizing cells:

  • RBCs are pink throughout
  • Neutrophils have a dark purple lobed nucleus
  • Lymphocytes have a large dark purple nucleus that occupies most of the cell
  • Monocytes have a kidney-shaped dark purple nucleus
  • Eosinophils have pink granules
  • Basophils have purple granules

Below is an image of my Leishman’s staining where a monocyte and numerous RBC are visible: Leishman Stain Blog

Basophils have purple granules In my sample, a monocyte is visible among numerous RBC.